The ubiquitin proteasome system (UPS) plays a central role in protein homeostasis through ubiquitination and degradation of substrate proteins. General inhibitors of the proteasome have proven effective in cancer therapy (Weathington, 2014), and thus there is great interest in developing specific inhibitors of UPS enzymes to explore their biological functions and to provide paths to more specific therapeutics. The central player in the UPS is ubiquitin (Ub), a highly conserved 76-residue protein. Ub is covalently attached to protein substrates through sequential action of ubiquitin activating (E1), ubiquitin conjugating (E2), and ubiquitin ligating (E3) enzymes. E3 ligases bind protein substrates and thus dictate specificity of ubiquitination.
E3 ligases constitute the largest class of UPS enzymes, with more than 600 members encoded by the human genome, and are divided into two major classes: a small, well-characterized class of approximately 30 HECT E3 ligases and a much larger, but less-characterized class of hundreds of RING E3 ligases and structurally related variants (Bhowmick). HECT E3 ligases form transient thioester linkages with Ub before transferring it to substrates, while RING ligases serve as adaptors to recruit Ub-charged E2 enzymes to substrates for Ub transfer. The archetype for the RING class is the multi-subunit Skp1-Cul1-F-box (SCF) complex family, which contains 69 members in humans (Jin, 2004). The SCF enzyme complexes are composed of constant Rbx1, Cul1, and Skp1 subunits and a variable F-box protein that binds substrates and dictates specificity (FIG. 1A). Rbx1, the RING protein that recruits the E2 enzyme, binds the scaffold protein Cul1, which in turn binds Skp1, an adaptor for F-box proteins. F-box proteins are variable in domain composition but share a common F-box domain that binds Skp1. F-box proteins are subdivided into three subfamilies based on the structure of their substrate binding domains including WD40, LRR, and other domains, referred to as the Fbw, Fbl, and Fbo subfamilies, respectively (Jin, 2004). Numerous F-box proteins are involved in processes relevant to tumorigenesis, including cell proliferation, cell cycle progression, and apoptosis, suggesting that these proteins may be targets for cancer treatment (Wang, 2014).